Identification of three separate guanine nucleotide-binding proteins that interact with the delta-opioid receptor in NG108-15 neuroblastoma x glioma hybrid cells.
Identification of three separate guanine nucleotide-binding proteins that interact with the delta-opioid receptor in NG108-15 neuroblastoma x glioma hybrid cells.
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发表时间：
1992-05
影响因子：
3.6
通讯作者：
S. Roerig;H. Loh;P. Law
中科院分区：
文献类型：
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作者：
S. Roerig;H. Loh;P. Law
在神经母细胞瘤与胶质瘤NG108-15杂交细胞的细胞膜上，免疫鉴定了5个独立的鸟嘌呤核苷酸结合蛋白（G蛋白）。这些α亚基蛋白是Gi2 α、Gi3 α的两个同工型和Go α的两个同工型。利用霍乱毒素（CTX）诱导的adp核糖基化和对各种G蛋白α亚基的抗血清选择性，鉴定了与这些膜上的δ -阿片受体相互作用的G蛋白。在阿片受体激动剂存在的情况下，CTX诱导[32P] adp核糖结合到三种百日咳毒素底物中。利用针对G α亚基肽序列产生的抗血清，这三种百日咳毒素底物被鉴定为Gi2 α、Go2 α和一种尚未被鉴定的Gi3 α的异构体。通过观察δ受体激动剂D-Ala2-D-Leu5-enkephalin （DA-DLE）和D-Pen2-D-Pen5-enkephalin以及非选择性激动剂艾托啡和布雷马嗪具有活性，而mu激动剂PL017和kappa激动剂U-50-488H则没有这种活性，证明ctx诱导的标记是通过δ -阿片受体介导的。DADLE诱导的三种底物的结合是剂量依赖性的，EC50（95%置信区间）值在12（3-52）至183 (65-520)nM之间，而该激动剂的Kd值为10 +/- 1.5 nM，该剂量对腺苷酸环化酶活性产生最大的抑制作用。此外，用百日咳毒素预处理细胞或用拮抗剂纳洛酮处理细胞膜可阻断DADLE诱导的掺入。adp -核糖在三种底物中的掺入减少了35-83%，而在这些膜中，这些受体被DADLE慢性治疗下调。因此，一种阿片受体类型可以与三种不同的G蛋白相互作用。
Five separate guanine nucleotide-binding proteins (G proteins) were immunologically identified in membranes from neuroblastoma x glioma NG108-15 hybrid cells. These alpha subunit proteins were Gi2 alpha, two isoforms of Gi3 alpha, and two isoforms of Go alpha. The G proteins that interacted with delta-opioid receptors in these membranes were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for various G protein alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into three pertussis toxin substrates. Using antisera generated against peptide sequences from G alpha subunits, these three pertussis toxin substrates were identified as Gi2 alpha, Go2 alpha, and one isoform of Gi3 alpha, which has yet to be identified. This CTX-induced labeling was demonstrated to be mediated via the delta-opioid receptor in these hybrid cells by the observation that delta agonists D-Ala2-D-Leu5-enkephalin (DA-DLE) and D-Pen2-D-Pen5-enkephalin, as well as the nonselective agonists etorphine and bremazocine, were active, but the mu agonist PL017 and the kappa agonist U-50-488H did not show this activity. This incorporation into all three substrates induced by DADLE was dose dependent, with EC50 (95% confidence interval) values ranging from 12 (3-52) to 183 (65-520) nM, which compared with the Kd value of 10 +/- 1.5 nM for this agonist, a dose that produces maximal inhibition of adenylate cyclase activity. Furthermore, pretreatment of the cells with pertussis toxin or treatment of the membranes with the antagonist naloxone blocked the incorporation induced by DADLE. Incorporation of [32P]ADP-ribose into all three substrates decreased 35-83% in membranes in which the receptors had been down-regulated by chronic treatment of the cells with DADLE. Thus, a single opioid receptor type can interact with three separate G proteins.